One of the major challenges in the era of microbial genomics is to experimentally establish connections between genome data and gene function, physiology, behavior and pathogenicity. Treponema denticola, an opportunistic pathogen of gingival mucosal tissue, offers a range of targets for research into microbe-host interactions, microbial physiology and molecular evolution. While pathogenic behaviors and disease associations of oral spirochetes are clearly distinct from those of T. pallidum, T. denticola is of high interest as a model for studying physiology, biosynthetic pathways and microbe-host interactions in spirochetal diseases including syphilis. This is due both to physiological similarities between Td and T. pallidum and to the fact that T. pallidum genetic studies remain impracticable without an in vitro culture system. This project will develop and demonstrate significant advances in methods for genetic manipulation of cultivable spirochetes of the genus Treponema that will jump-start Treponema genetic research, thus providing opportunities to rationally address novel biological questions, particularly in uncultivable spirochetes. We propose a T. denticola-specific adaptation of counter selection mutagenesis coupled with knockouts of restriction-modification systems as key features to facilitate construction of T. denticola strains with (1) greatly increased ability to take up and incorporate heterologous DNA's, and (2) an expanded range of possible mutant types. Building on proof of concept studies primarily conducted by our group, results of this work will both facilitate in-depth molecular analysis of T. denticola and establish the utility of T. denticola as a model organism for molecular analysis of agents of other treponemal infections, particularly T. pallidum.